Multiresidue Analysis of Veterinary Drugs in Bovine Liver by LC/MS/MS

Significance of the Topic

The accurate detection of multiple veterinary drug residues in fatty food matrices such as bovine liver is critical for food safety and regulatory compliance. High lipid content interferes with chromatographic and mass spectrometric analysis, reducing sensitivity and precision. The use of a selective lipid‐removal sorbent simplifies sample preparation, minimizes matrix effects, and supports multiresidue monitoring programs.

Objectives and Study Overview

This study evaluated Agilent’s Bond Elut Enhanced Matrix Removal—Lipid (EMR—Lipid) dispersive solid‐phase extraction (dSPE) for cleanup of acidified acetonitrile liver extracts prior to LC/MS/MS analysis of 30 representative veterinary drugs. Key goals were to compare lipid‐removal efficiency and analyte recovery against traditional C18 and zirconia sorbents, optimize sample preparation conditions, and validate a robust multiresidue method.

Methodology and Instrumentation

Sample preparation comprised:

• Weigh 2 g homogenized bovine liver, spike internal standard, and extract with 10 mL acetonitrile containing 5% formic acid.
• Centrifuge and transfer 5 mL supernatant to EMR—Lipid dSPE tube with 5 mM ammonium acetate activation buffer; vortex and centrifuge.
• Perform a polish step by adding MgSO₄/NaCl salt mix in an EMR—Lipid polish tube; vortex, centrifuge, then dilute with water for injection.

Instrumentation included an Agilent 1290 Infinity UHPLC coupled to a 6490 triple‐quadrupole mass spectrometer with Jet Stream electrospray. A Poroshell C18 column and a binary gradient of 0.1% formic acid in water/acetonitrile at 0.3 mL/min achieved separation of acidic, neutral, and basic analytes.

Main Results and Discussion

The EMR—Lipid cleanup removed 56% of coextractives by weight—significantly more than zirconia (50%) or C18 (35%). Postcolumn infusion experiments demonstrated dramatically reduced matrix suppression/enhancement. Optimization showed that 5% formic acid and 5 mM ammonium acetate buffer enhanced analyte recoveries and calibration linearity. A simplified non‐polish protocol improved tetracycline recoveries to >85%, though the polish step delivered acceptable precision. Compared to QuEChERS workflows with C18 or zirconia dSPE, EMR—Lipid delivered superior recoveries (>70% for 28/30 analytes) and RSDs below 10% for most compounds. Calibration curves were linear (R²>0.99) over 5–1,000 ng/g (Group 1) and 1–200 ng/g (Group 2). Method validation across four concentration levels achieved 70–120% accuracy for 93% of analytes with RSDs typically <8%.

Benefits and Practical Applications

• Efficient, selective lipid removal improves data quality and instrument uptime.
• Simplified workflow reduces sample handling time and risk of analyte loss.
• Suitable for high‐throughput laboratories monitoring multiclass residues in fatty tissues.

Future Trends and Potential Applications

Further studies will explore EMR—Lipid’s performance in other complex matrices (milk, fish, egg) and extend to additional analyte classes, including emerging contaminants. Integration with automated sample preparation platforms and high‐resolution mass spectrometry may broaden scope and throughput.

Conclusion

The EMR—Lipid dSPE workflow combined with protein precipitation offers a fast, robust, and reproducible method for multiresidue veterinary drug analysis in bovine liver. Superior matrix cleanup and consistent recoveries facilitate reliable LC/MS/MS quantitation in high‐fat samples.

References

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• SANCO/12571/2013 Guidance on QC and validation procedures for pesticide residues analysis.